Platelet -granules constitute the major rapidly releasable reservoir of thrombospondin-1 in higher animals, but its physiological function in platelets has remained elusive. We found that endogenous thrombospondin-1 is necessary for platelet aggregation in vitro in the presence of physiological levels of nitric oxide (NO). Exogenous NO or elevation of cGMP delays thrombin-induced platelets aggregation, and exogenous thrombospondin-1 reverses this delay. Thrombospondin-1 null murine platelets fail to aggregate in response to thrombin in the presence of exogenous NO or 8Br-cGMP. At physiological concentrations of the NO synthase substrate arginine, thrombospondin-1 null platelets have elevated basal cGMP. Ligation of CD36 or CD47 is sufficient to block NO-induced cGMP accumulation and mimic the effect of thrombospondin-1 on aggregation. Exogenous thrombospondin-1 also reverses the suppression by NO of IIb/3 integrin-mediated platelet adhesion on immobilized fibrinogen, mediated in part by increased GTP loading of Rap1. Thrombospondin-1 also inhibits cGMP-mediated activation of cGMP-dependent protein kinase and thereby prevents phosphorylation of VASP. Thus, release of thrombospondin-1 from -granules during activation provides positive feedback to promote efficient platelet aggregation and adhesion by overcoming the anti-thrombotic activity of physiological NO. Studies using TSP1, CD36, and CD47 null mice have extended the role of TSP1 as an antagonist of NO/cGMP signaling to limiting the survival of full thickness skin grafts and recovery from ischemia/reperfusion injury in the liver. Both injuries show increased recovery in mice lacking TSP1 or its receptor CD47, and treatment of wild type using antibodies or antisense oligonucleotides that block TSP1 binding to CD47 improve recovery from the same injuries. The therapeutic efficacy of these agents was extended to a large mammal that has a vascular anatomy more closely resembling humans. A fixed soft tissue ischemia model was established in the Yucatan miniature pig. Treatment with a TSP1 antibody or a CD47 antisense morpholino significantly enhanced survival of ischemic tissues in these animals. Survival of ischemic injury is known to decrease with age. Because cancer is primarily a disease of aging, this limits the ability of surgeons to successfully reconstruct soft tissues following excision of tumors. We recently found that TSP1 plays an important role in the age-dependent decrease in resistance to ischemic injury. Mice lacking either TSP1 or CD47 show minimal loss of their resistance to ischemic injury with age and increased preservation of tissue perfusion immediately following injury. Treatment of wild type mice using therapeutic agents that decrease CD47 or enhance NO reverse the deleterious effects of age and diet-induced vasculopathy and result in significantly increased tissue survival in models of ischemia. Therefore, drugs that limit TSP1/CD47 regulation of blood flow could improve outcomes from surgical interventions in the elderly and ameliorate vascular complications attendant to ageing. In solid tumors, NO is generally acknowledged to mediate angiogenic responses to several growth factors. This contrasts with conflicting evidence that NO can acutely increase tumor perfusion through local vasodilation or diminish perfusion by preferential relaxation of peripheral vascular beds outside of the tumor. Because TSP1 is an important physiological antagonist of NO in vascular cells we examined whether, in addition to inhibiting tumor angiogenesis, TSP1 can acutely regulate tumor blood flow. We assessed this activity of TSP1 in the context of perfusion responses to NO as a vasodilator and epinephrine as a vasoconstrictor. NO treatment of wild type and TSP1 null mice decreased perfusion of a syngeneic melanoma, while epinephrine transiently increased tumor perfusion. Acute vasoactive responses were also independent of the level of tumor-expressed TSP1 in a melanoma xenograft, but recovery of basal perfusion was modulated by TSP1 expression. In contrast, over-expression of truncated TSP1 lacking part of its CD47 binding domain lacked this modulating activity. These data indicate that TSP1 primarily regulates long term vascular responses in tumors, in part because the tumor vasculature has a limited capacity to acutely respond to vasoactive agents. Radiation injury is another stress that is relevant to cancer therapy. Agents are needed to increase sensitivity of tumors or to protect healthy tissue from radiation injury. We found that soft tissues in thrombospondin-1 null mice are remarkably resistant to radiation injury. Twelve hours after 25 Gy hindlimb irradiation, thrombospondin-1 null mice show significantly less cell death in muscle and bone marrow. Two months following irradiation, skin and muscle units in the null mice show minimal histological evidence of radiation injury and near full retention of mitochondrial function. Tissue perfusion and acute vascular responses to NO are also preserved in irradiated thrombospondin-1 null hindlimbs. The role of thrombospondin-1 in radiosensitization is specific in that thrombospondin-2 null mice were not protected. However, mice lacking the thrombospondin-1 receptor CD47 showed similar radioresistance as thrombospondin-1 null mice. Thrombospondin-1- and CD47-dependent radiosensitization is cell autonomous because vascular cells isolated from the respective null mice showed dramatically increased survival and improved proliferative capacity following irradiation in vitro.
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